Project Summary The interplay between cellular adhesion and cellular signaling is essential for the development of all organs such as the brain, and for the functioning of systems such as the nervous systems. Teneurins (TEN1-4) are a poorly understood family that mediates intercellular communication. They have essential roles in embryonic development and neural circuit-wiring; and are linked to numerous human diseases including neurological disorders and cancers. TENs are type-II membrane proteins with large C-terminal extracellular regions (ECR) that majorly exhibit no identifiable domains. The ECR mediates trans-cellular heterophilic interaction of TENs with Latrophilins(LPHN1-3), a family of G-Protein Coupled Receptors; to regulate synapse function. The ECR also mediates trans-cellular homophilic interaction of TENs with themselves to instruct neural circuit-wiring. However, the molecular mechanisms underlying TEN action remains poorly understood majorly due to the lack of structural information on the ECR. We recently laid the groundwork by determining the high-resolution cryo-EM structure of the TEN2 ECR and revealed a surprising homology to bacterial Tc-toxins. We also showed that an alternatively spliced insert acts as a switch to regulate LPHN binding and other TEN functions such as synapse formation. The ultimate goal of the research proposed in this application is to understand the mechanical details of various TEN functions that are mediated by its ECR. We propose three Specific Aims that are based on the major unknowns in TEN function and a Follow-up Aim to perform structure/function relationship studies: First, we aim to understand the molecular details of the TEN/LPHN interaction. Second, we aim to understand the molecular determinants for the trans-homodimerization of TEN. Third, we aim to reveal whether TEN functions via autoproteolysis similar to bacterial toxins. Then, we aim to use the information from the first three aims to study TEN function in synapse formation assays. This research has a multi-disciplinary approach where the structural and functional data performed in the PI's lab range from electron microscopy, biophysical and biochemical methods, neuronal assays to cell-biology and is complemented by the expertise provided or performed by the laboratories of close collaborators. The proposed experiments will build on exciting results, including the very unusual TEN2 structure, surprising involvement of alternative splicing in TEN function, key advances in the purification of all needed TEN fragments, and the observation of proteolytic products. We expect that this research will provide critical insights into the mechanistic details of TEN function, helping to establish novel principles on intercellular communication that are vital for numerous cellular functions.